Much of the progress of modern medicine, environmental protection and materials development and production can be assigned to the advances in chemical analytical systems that permit the separation, identification, characterization and quantification of various chemical moieties. Separating the components of a sample mixture is one of the key processes in an analytical procedure. Components so separated can be individually identified and quantified.
Electrophoresis separates chemical components of a sample on the basis of their component-specific migration rate in an applied electric field along a separation path. A large number of biological or chemical molecules/macromolecules are charged in aqueous solutions at appropriate pH values. For example proteins are amphoteric and their charge varies from positive to negative, depending on the protein and the pH-value. Electrophoretic separation methods are based on the different mobilities of the individual components of the sample in an electrophoresis medium when an electric field is applied. The electrophoretic mobility of each individual component is the velocity it attains for a given external electric field and corresponds to the ratio between charge of the component and its frictional drag. The sample is applied as a narrow zone in the separation path and electrophoretic migration is started by the application of the electric field. Each component migrates at its specific rate along the separation path and forms a so-called band. Each band can be characterized by a peak, where the component concentration is maximal, and a width, being the distance between points on either side of the peak where the concentration falls below a certain threshold value. The width of the sample zone produced by the application of the sample and subsequent diffusion contribute to the widths of the electrophoretic bands.
Generally the following performance criteria are important for electrophoretic separation methods.                Resolution. Two components are usually separated when their migration distances differ more than the sum of the half-widths of their electrophoretic bands.        Detection limit. The lower detection limit of a component in a given sample decreases with the width of its separated band. The narrower this band is, the higher the concentration of the separated compound in the band will be and the better its detection can be.        Speed and throughput. Throughput is proportional to the number of parallel separations, for example in an array of separation channels, and inversely proportional to the time for one separation.        Convenience, practicability and cost-effectiveness. Miniaturization of CE, for example in chip format, can provide the fulfilling of these criteria (REF). These criteria are important for point-of-care and point-of-use applications of CE.        
Minimizing the bandwidth of the separated components essentially contributes to fulfil the criteria of high resolution and low detection limit. Two main factors contribute to the width of the electrophoretic band, the width of the applied sample zone and its broadening due to diffusion during electrophoresis. The volume of the applied sample solution which is required for the detection of the components of interest determines the width of the sample zone. The majority of practical samples, for example in medicine or environmental and bioprocess monitoring, are concentration-limited, i.e. samples where the amount of the component to be analyzed is limited by its concentration in the sample and not by the available sample volume.
Usually high-resolution and sensitive electrophoretic separations of concentration-limited samples require a (pre-)concentration step. One problem underlying this invention is the convenient realization of the concentration step in a miniaturized capillary electrophoresis device and method. Hitherto concentration steps were achieved by discontinuous electrophoretic media in the separation channel, e.g. in polyacrylamide disc electrophoresis where a sample is concentrated at the boundary between different gels and buffers (REF). Discontinuous electrophoretic media in capillary electrophoresis can only be realized by cumbersome and tedious techniques, and only once for each sample.
Capillary electrophoresis is a widely used, well established analytical separation technique in bioscience, in pharmaceutical, environmental, food and other chemical analyses. In capillary electrophoresis a sample is injected into a carrier medium, where the individual components migrate at different rates in an applied external electrical field along the separation path, with the result that the sample is separated into its components. The separated components usually are determined by a detector connected to the capillary separating path (e.g. optical detector for fluorescence detection). Conventional high performance capillary electrophoresis (HPCE) instruments apply fused silica capillaries of 25–75 μm inner diameter and up to 1 m length and apply voltages of 10–30 kV. Special surface modifications, such as coatings, are used to improve separation performance. A typical separation procedure takes about 5–30 minutes.
Microfabricated, miniaturized capillary electrophoresis chips were introduced in 1992, see A. Manz, D. J. Harrison, E. M. J. Verpoorte, J. C. Fettinger, A. Paulus, H. Ludi, H. M. Widmer, J. Chromatogr. 593 (1992) S. 253. Separations e.g. of fluorescent dyes (see D. J. Harrison, A. Manz, Z. Fan, H. Ludi, H. M. Widmer, Anal. Chem. 64 (1992) S.1926 and see also S. C. Jacobson, R. Hergenroeder, L. B. Koutny, R. J. Warmack, J. M. Ramsey, Anal. Chem. 66 (1994) S.1107), fluorescently labeled amino acids (see D. J. Harrison, K. Fluri, K. Seiler, Z. Fan, C. S. Effenhauser, A. Manz, Science 161 (1993) S.895; C. S. Effenhauser, A. Manz, H. M. Widmer, Anal. Chem. 65 (1993) S.2637; S. C. Jacobson, R. Hergenroeder, A. W. Moore, J. M. Ramsey, Anal. Chem. 66 (1994) S.4127) and metal ion complexes (S. C. Jacobson, A. W. Moore, J. M. Ramsey, Anal. Chem. 67 (1995) S.2059) have shown that the separation speed of such devices can be increased by about more than one order of magnitude.
Therefore miniaturized capillary electrophoresis chips have been used for rapid analysis of biological samples, e.g. DNA restriction fragments (A. T. Wooley, R. A. Mathies, Proc. Natl. Acad. Sci. USA 91(1994) S.11348; S. C. Jacobson, J. M. Ramsey, Anal. Chem. 68 (1996) S.720), DNA sequencing fragments (A. T. Wooley, R. A. Mathies, Anal. Chem. 67 (1995) S.3676), PCR products (see S. C. Jacobson, J. M. Ramsey, Anal. Chem. 68 (1996) S.720) and short oligonucleotides (C. S. Effenhauser, A. Paulus, A. Manz, H. M. Widmer, Anal. Chem. 66 (1994) S.2949).
Also miniaturization aims at the integration of electrophoretic separation into total-analysis-systems (μTAS) or lab-on-a-chip solutions.
Miniaturization of capillary electrophoresis promises besides aspects of high separation performance also economic aspects like cost-effective production as well as the possibility of operation by untrained personnel including highly reliable procedures.
An electrophoretic separating device as well an electrophoretic separating method is disclosed in U.S. Pat. No. 5,296,114. The device comprises a separating channel which is constructed basically in form of a closed loop for example in form of a square. At each corner of the square at least one opening is provided having an electrode. A sample to be separated is introduced via a feed opening into said separating channel which is filled with an electrolytic carrier medium. The dissolved sample is moved with the aid of an electrical field through the channel, which is provided by the electrodes in the regions of the openings and is separated into individual components by the electrical field. The electrical field is generated in the channel by connecting electrodes in the region of the openings to different potentials of a voltage source. Due to the fact that the separating channel is square no dense packing of a multitude of separating channels isn t possible to arrange onto one carrier substrate.
In summary, a miniaturized CE separation device which can conveniently achieve a narrow bandwidth of the separated components from a concentration-limited sample has not been disclosed yet and hence is the objective of the described invention.